Yequan Wang

Differential expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in anterior cruciate ligament and medial collateral ligament fibroblasts after a mechanical injury: Involvement of the p65 subunit of NF-κB

The anterior cruciate ligament (ACL) is known to have a poor healing ability, especially in comparison with the medial collateral ligament, which can heal relatively well. In this study, we detected significant increases in the mRNA levels of multiple matrix metalloproteinases (MMPs) (MMP‐1, ‐2, ‐7, ‐9, ‐11, ‐14, ‐17, ‐21, ‐23A, ‐24, ‐25, ‐27, and ‐28) and tissue inhibitors of metalloproteinases (TIMPs) (TIMP‐1, ‐2, ‐3, and ‐4) in ACL fibroblasts after an in vitro injury with an equi‐biaxial stretch chamber. However, only some MMPs (MMP‐7, ‐9, ‐14, ‐21, and ‐24) showed increases in injured medial collateral ligament fibroblasts, and to a much lesser degree than that observed in the injured ACL fibroblasts. Zymography revealed a 6.3‐fold increase of MMP‐2 activity in injured ACL but not medial collateral ligament fibroblasts, which agrees with the global MMP activities assay. Bay‐11 and curcumin can significantly decrease MMP‐2 activities to 13% and 29% in injured ACL fibroblasts, respectively, which implies the involvement of p65 subunits of nuclear factor κB and AP‐1 pathways. Furthermore, Bay‐11 can decrease the global MMP activity released from injured ACL fibroblasts in a dose‐dependent manner. In summary, the differential expression and activities of MMPs might help to explain the poor healing ability of ACL, and the p65 subunit of nuclear factor κB might be a potential target to facilitate the ACL repair.

Combined effects of TNF-α, IL-1β, and HIF-1α on MMP-2 production in ACL fibroblasts under mechanical stretch: An in vitro study

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra‐articular joint, are very complex after injury. Injury to intra‐articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro‐inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix‐metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP‐2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor‐1α (HIF‐1α) on induction of MMP‐2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF‐α and IL‐1β elevate the activity of MMP‐2 in a dose‐ and time‐dependent manner. In addition, mechanical stretch further enhances the MMP‐2 protein levels with TNF‐α, IL‐1β, and their mixture. CoCl2‐induced HIF‐1α (100 and 500 µM) also increases the levels and activity of MMP‐2. Mechanical stretch has a strong additional effect on MMP‐2 production with HIF‐1α. Our results conclude that mechanical injury, HIF‐1α and inflammatory factors collectively induce increased MMP‐2 production in ACL fibroblasts, which was inhibited by NF‐κB pathway inhibitor (Bay‐11‐7082).

Expression of MMPs and TIMPs Family in Human ACL and MCL Fibroblasts

The human ACL (anterior cruciate ligament) is susceptible to injury but has poor healing response, whereas an injured MCL (medial collateral ligament) can be repaired relatively well. Since MMPs (matrix metalloproteases) and TIMPs (tissue inhibitor of metalloproteases) are involved in this tissue remodeling process, investigation of different response of MMPs and TIMPs family in ACL and MCL fibroblasts might lead to understanding the differential matrix remodeling process as well as their different healing ability. The first step would be determination of whether these tissue remodeling effectors are present in ligaments. In this study, we designed primers for real-time RT-PCR and determined the expression of MMPs and TIMPs family in ACL and MCL fibroblasts with synovium as a positive control. Semiquantitative RT-PCR revealed that multiple MMPs and TIMPs expressed in human ACL and MCL fibroblasts except MMP-8, 10, 12, 13, 15, 16, 20, and 26. MMP-7 was present in MCL but not in ACL fibroblast. Quantitative real-time RT-PCR showed that mRNA levels of MMP-1, 2, 14, 17, 23A, and 23B and TIMP-4 are significantly higher in MCL than in ACL fibroblasts. However, MMP-3 is higher in ACL than in MCL fibroblasts. We conclude that numerous MMPs and TIMPs family members that are differentially expressed in ACL and MCL might be involved in the differential matrix remodeling process as well as the differential healing ability of ACL and MCL.

Differential response to CoCl2-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells

The adult human anterior cruciate ligament (ACL) has a poor functional healing response, whereas the medial collateral ligament (MCL) does not. The difference in intrinsic properties of these ligament cells can be due to their different response to their located microenvironment. Hypoxia is a key environmental regulator after ligament injury. In this study, we investigated the differential response of ACL and MCL fibroblasts to hypoxia on hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinase-2 (MMP-2) expression. Our results show that ACL cells responded to hypoxia by up-regulating the HIF-1α expression significantly as compared to MCL cells. We also observed that in MCL fibroblasts response to hypoxia resulted in increase in expression of VEGF as compared to ACL fibroblasts. After hypoxia treatment, mRNA and protein levels of MMP-2 increased in both ACL and MCL. Furthermore we found in ACL pro-MMP-2 was converted more into active form. However, hypoxia decreased the percentage of wound closure for both ligament cells and had a greater effect on ACL fibroblasts. These results demonstrate that ACL and MCL fibroblasts respond differently under the hypoxic conditions suggesting that these differences in intrinsic properties may contribute to their different healing responses and abilities.

Coordinated Expression of MMPs and TIMPs in Rat Knee Intra-Articular Tissues After ACL Injury

Anterior cruciate ligament (ACL) has poor healing ability and an injured ACL would induce the degeneration of other intra-articular connective tissues. However, the coordinated expression and activities of matrix metalloproteinase (MMPs) in intra-articular tissues induced by ACL rupture were poorly understood. With a rat ACL rotating injury model, we found that after ACL injury, the mRNA levels of MMP-13, TIMP-1, and CD147 were significantly elevated in ACL, posterior cruciate ligament (PCL), synovium, meniscus, and cartilage. Also, MMP-2 activity was also elevated significantly in a time-dependent manner in all intra-articular tissues. Synovium showed the most capability to release MMPs, whereas ACL showed the highest MMP-13/TIMP-1 ratio. Generic MMP activity assay and zymography showed time dependent elevation of MMP activities in synovial fluids (SF). We concluded that the ACL injury would induce a coordinated response of intra-articular tissues to express MMPs, TIMPs, and CD147. The MMP activities in the microenvironment in SF would accumulate, released by all the intra-articular tissues, which would contribute to the knee damage and degeneration induced by ACL injury.

MMPs and TIMPs Expression and the Anterior Cruciate Ligament Repair

Human anterior cruciate ligament (ACL) has poor healing ability after injury. Inflammation and accumulation of matrix metalloproteinases (MMPs) in synovial fluids may be involved in the process. Tissue inhibitor metalloproteinases (TIMPs) may be independent of MMPs inhibition. A rat ACL rotating injury apparatus was designed to mimic ACL injury. ACL showed the higher MMP-1/TIMP-1 ratio after injury. Our recently findings demonstrated that MMPs expression and activities elevated significantly in the joint after ACL injury. This maybe one of the important reasons why an injured ACL cannot be repaired.

Role of NF -κB in the Injury Induced MMP Expression and Activities in ACL

The anterior cruciate ligament (ACL) of the human knee plays an important role in controlling and stabilizing the knee joint, but has poor healing response, especially in comparison with the medial collateral ligament (MCL), which can heal relatively well. In this study, we detected significant increases in the mRNA levels of multiple matrix metalloproteinases (MMPs) (MMP-1, 2, 7, 9, 11, 14, 17, 21, 23A, 24,25,27 and 28) in ACL fibroblasts after in vitro injury with equi-biaxial stretch chamber. However, only some MMPs (MMP-7, 9,14, 21 and 24) showed increases in injured MCL fibroblasts, and to a much lesser degree than observed in the injured ACL fibroblasts. Zymography revealed 6.3-folds increase of MMP-2 activity in injured ACL but not MCL fibroblasts which parallel with the global MMP activities assay. Bay-11 and curcumin can significantly decrease MMP-2 activities to 13% and 29% in injured ACL fibroblasts respectively, which implies the involvement of p65 subunits of NF-κB and AP-1 pathways. Furthermore, Bay-11 can decrease the global MMP activity released from injured ACL fibroblasts in a dose-dependent manner. In summary, the differential expression and activities of MMPs might help to explain poor healing ability of ACL and p65 subunit of NF-κB might be a potential target to facilitate the ACL repair.